28th June 2018

Peptidome

The set of peptides presented by class I MHC molecules at the cell surface. Predominantly 9 amino acids in length.

Predominantly 9 amino acids in length.

Hypotheses for the role of MHC I in skin sensitisation

  • Sensitiser-modified peptides presented by MHC I directly stimulate the immune system.
  • Sensitiser modification of proteins alters the MHC I presented repertoire of peptides.

HaCaTs and DNCB

  • HaCaT cell line is a spontaneously transformed keratinocyte.
  • Homozygous HLA-A*31:01, HLA-B*40:01 / HLA-B*51:01, HLA-C*03 / HLA-C*15
  • 2,4-Dinitrochlorobenzene (DNCB) is our model sensitiser.
  • DNCB modifies 0.12% Lys, 0.03% Cys and < 0.03% Tyr, His and Arg.

HaCaT peptidome

Wildtype

HaCaT N-mer peptide counts

HaCaT N-mer peptide frequency

Immune epitope database peptides

HaCaT predicted peptides

DNCB modified peptides

Peptide Length Replicate UNIPROT ID
AETEC(+166.00)RYAL 9 DNCB 1A P13646
RFC(+166.00)PFAERTR 10 DNCB 1A P78417
RFC(+166.00)PFAERTR 10 DNCB 1B P78417
AETEC(+166.00)RYAL 9 DNCB 1B P13646
AETEC(+166.00)RYAL 9 DNCB 3A P13646
RFC(+166.00)PFAERTR 10 DNCB 3A P78417
AETEC(+166.00)RYAL 9 DNCB 3B P13646

HLA-B*40:01 9-mer AETEC(+DNP)RYAL

Keratin, type I cytoskeletal 13

HLA-A*31:01 10-mer RFC(+DNP)PFAERTR

Glutathione S-transferase omega-1

Glutathione S-transferase omega-1

Next steps

  • HLA-B*40:01, 9mer, AETECRYAL, Keratin, type I cytoskeletal 13
  • HLA-A*31:01, 10mer, RFCPFAERTR, Glutathione S-transferase omega-1
  • HLA-A*02:01, 9mer, YSMRFCPFA, Glutathione S-transferase omega-1

Erika's observes DNCB modified tryptic peptides :

  • MFAPTK(+DNP)TWR, HLA-A*31:01, 9mer, MFAPTKTWR, 60S ribosomal protein L4
  • YFDK(+DNP)ASYR, HLA-A*31:01, 9mer, KYFDKASYR, Transketolase

Erika also observed MCI modified Glutathione S-transferase P:

  • PPYTVVY(+MCI)FPVR, HLA-A*31:01 9-mer, VVYFPVRGR

HaCaT peptidome

HLA-A*02:01 transfectant

HLA-A2 transfectant N-mer peptide counts

HLA-A2 transfectant N-mer peptide frequency

Immune epitope database peptides

Machine Learning classified peptides

Predicted HLA-A2 peptides

P13646: Keratin type I cytoskeletal 13, only seen as unmodified peptides

Peptide Length Replicate UNIPROT ID
MLLDIKTRL 9 Control 1 P13646
MLLDIKTRL 9 Control 2 P13646
RLASYLEKV 9 Control 2 P13646
MLLDIKTRL 9 Control 3 P13646
MLLDIKTRL 9 DNCB 1 P13646
MLLDIKTRL 9 DNCB 2 P13646

Glutathione S-transferase Mu 4

HLA-A31 peptide also seen in WT assays

Peptide Length Allotype UNIPROT ID
FLGKRPWFV 9 HLA-A2 Q03013
RFLPKPLYTR 10 HLA-A31 Q03013

DNCB modified HLA-A2 peptides

P13647 : Keratin, type II cytoskeletal 5

P13489 : Ribonuclease inhibitor

Peptide Length Replicate UNIPROT ID
KLLEGEEC(+166.00)RL 10 DNCB 2 P13647
GLLDPQC(+166.00)RL 9 DNCB 2 P13489
KLLEGEEC(+166.00)RL 10 DNCB 3 P13647

HLA-A*02:01 9-mer, GLLDPQC(+DNP)RL

Ribonuclease inhibitor

Ribonuclease inhibitor

HLA-A*02:01 10-mer, KLLEGEEC(+DNP)RL

Keratin, type II cytoskeletal 5

Story so far

Evidence for direct presentation of sensitiser modified peptides:

  • HLA peptides identified by mass spectrometry.
  • Peptides predicted to be strong binders.
  • Observing modified peptides from several proteins is not inconsistent with the ability of DNCB to induce contact hypersensitivity in all immunocompetent individuals.

Evidence for sensitiser effect on cellular detoxification mechanism:

  • Modification of Glutathione S-transferase omega-1.

Central questions

Are these HLA peptides real and immunogenic?

  • Synthesise peptides to verify spectra.
  • Test immunogenecity on patient samples.

Why do we only observe cysteine modifications for HLA peptides?

What is DNCB doing to the proteome?

  • Process and analyse protein turnover data.
  • Integrate peptidome data with Sergio and Erika's data.

Does DNCB modulate specific pathways?

  • Look for further evidence of presentation of peptides from proteins associated with oxidative stress.